title: Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells.
abstract: Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.
authors.affiliation: Department of Genetics, Biology and Biochemistry, University of Turin, via Santena 5 bis, 10126 Turin, Italy. antonella.pantaleo@unito.it
authors.forename: Antonella
authors.initials: A
authors.lastname: Pantaleo
authors.forename: Emanuela
authors.initials: E
authors.lastname: Ferru
authors.forename: Giuliana
authors.initials: G
authors.lastname: Giribaldi
authors.forename: Franca
authors.initials: F
authors.lastname: Mannu
authors.forename: Franco
authors.initials: F
authors.lastname: Carta
authors.forename: Alessandro
authors.initials: A
authors.lastname: Matte
authors.forename: Lucia
authors.initials: L
authors.lastname: de Franceschi
authors.forename: Franco
authors.initials: F
authors.lastname: Turrini
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D000906:Antibodies / Q000378:metabolism / Q000502:physiology; D003958:Diamide / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism / Q000473:pathology*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000097:blood / Q000378:metabolism / Q000473:pathology*; D006031:Glycosylation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D000906:Antibodies; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D013439:Sulfhydryl Reagents; D003958:Diamide; D014443:Tyrosine; D005954:Glucosephosphate Dehydrogenase; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase